tbuckley/cabot-search huggingface dataset:
O08 Using single-cell transcriptomics to characterize early mechanisms of psoriasis resolution
Authors: Luc Francis, Daniel McCluskey, Clarisse  Ganier, Xinyi Du-Harpur, David Baudry, Pawan  Dhami, Jonathon Barker, Catherine Smith, Francesca  Capon, Satveer Mahil
Journal: British Journal of Dermatology (2023)

Abstract The use of biological drugs targeting the interleukin (IL)-23/IL-17 axis has greatly improved outcomes in people with psoriasis, with an increasing number achieving clear skin (‘remission’). However, the early mechanisms of biologic-induced psoriasis resolution are yet to be fully explored. Longitudinal sampling of resolving disease tissue provides the opportunity to uncover the dynamic cellular and molecular events triggered by these powerful treatments. Identifying the early sequence of events and key drivers underpinning psoriasis resolution may help to guide future treatment strategies. We performed single-cell RNA sequencing (scRNA-seq) on skin biopsies from five individuals with psoriasis who achieved remission upon treatment with the exemplar IL23p19 inhibitor biologic risankizumab. We profiled whole skin at baseline, and at days 3 and 14 of treatment. We filtered, integrated, scaled and normalized the scRNA-seq data set using R. Ingenuity pathway analysis and the CellChat R package were used to explore canonical pathways and receptor–ligand interactions, respectively. RNA in situ hybridization (RNA ISH) was used to identify the spatial context of cell populations of interest using three independently ascertained patients, sampled at days 0 and 14 of risankizumab treatment. The final scRNA-seq data set comprised 164 741 cells We identified 39 clusters in whole skin, including keratinocyte, fibroblast, myeloid cell and T-cell subsets. We found marked differences in transcript levels between day 0 and day 3 of biological treatment using differential gene expression analysis, with the largest number of modulated genes observed among fibroblast and myeloid cell types. We identified a novel fibroblast population which was reduced in relative abundance at day 14 of treatment in lesional psoriasis skin and absent in nonlesional skin. Pathway analysis of this fibroblast population showed an association with inflammatory signalling, and receptor–ligand interaction analysis revealed proinflammatory cytokine signalling to differentiated keratinocytes. The presence of this fibroblast population in psoriasis skin was validated by deconvolution of independent, publicly available bulk RNA sequencing data sets. Finally, we confirmed the localization of this fibroblast population in the upper dermis of psoriasis skin and the reduced abundance after 14 days of risankizumab treatment using RNA ISH of lesional skin from three independently ascertained patients. Our findings provide a high-resolution atlas of the early changes in the skin microenvironment of resolving psoriasis. We identify a response in a novel fibroblast population in the early stages of biological treatment. We are currently exploring the role of this fibroblast population as a driver cell type in psoriasis resolution.
P31 Dissecting the epigenetic and transcriptional regulators in keloid scars
Authors: Stavroula Tekkela, Elena M Drudi, Tanya Shaw, Mike Philpott, Edel O’Toole, Emanuel Rognoni
Journal: British Journal of Dermatology (2023)

Abstract Keloids are a type of fibroproliferative scar that progressively grow beyond the original wound site. Currently, there are no treatments to halt or revert keloid scarring, emphasizing the great unmet clinical need and incomplete mechanistic understanding. They occur due to genetic, epigenetic and environmental abnormalities. However, the detailed molecular signatures that promote this pathogenic phenotype are still unknown. Fibroblasts are best characterized for their role in depositing and remodelling the extracellular matrix and presumed to be one of the main cells involved in keloid. This study investigates if keloid fibroblasts are epigenetically locked in an activated state, and if manipulation of this state can reverse the persistent keloid phenotype. To investigate the epigenetic and genetic changes in keloid fibroblasts, we performed ATACseq and RNAseq on a patient-matched set of keloid and normal fibroblasts. ATACseq is a high-throughput sequencing method that defines chromatin-accessible regions in which transcription factor binding sites can be mapped that can regulate gene expression. Differences in chromatin accessibility were found to be closely associated with gene expression differences in keloid fibroblasts. ATACseq analysis revealed accessible regions at genes that are implicated in pathways, including transcriptional regulation, embryonic development, calcium binding, collagen remodelling and apoptosis. Identified candidates driving keloid formation will be validated in keloid skin sections and functionally analysed in two- and three-dimensional culture models after pharmacological or genetic manipulation. These findings will support the development of fibroblast-targeted therapies for keloid scars and possibly other connective tissue disorders.
P18 KLICK syndrome is a hyperproliferative inflammatory skin disorder
Authors: Hannah Cherry, Maddy Parsons, Joe Rastrick, Jonathan Hardman-Smart, Paola DiMeglio, John McGrath
Journal: British Journal of Dermatology (2023)

Abstract Keratosis linearis with ichthyosis congenita and sclerosing keratoderma (KLICK) syndrome is an ultra-rare genodermatosis. Clinical manifestations include linear hyperkeratotic papules in the flexures, palmoplantar keratoderma and pseudoainhum. To date, all affected individuals carry a homozygous pathogenic variant, c.-95delC in POMP, which encodes for proteasome maturation protein. Disease pathophysiology is not well understood, resulting in nonspecific and limited treatment options. However, clinically, the phenotype shows erythema and inflammation, indicating that there may be uncharacterized inflammatory pathways in KLICK syndrome, similar to some other ichthyoses. The aim of this work was to characterize KLICK syndrome at the cellular and molecular level. A lesional skin biopsy was obtained, following informed consent, from a 32-year-old woman with KLICK syndrome. Sanger sequencing confirmed the presence of the homozygous single nucleotide deletion in the 5′ untranslated region in POMP. Skin architecture was assessed using immunostaining, which revealed dynamic proliferative keratinocytes, precocious expression of involucrin and loricrin, and retention of loricrin in the stratum corneum. Lymphocytic infiltrates in the upper dermis were suggestive of inflammation and imaging mass cytometry was used to visualize and identify the immune subsets present. RNA sequencing revealed dysregulated pathways relating to keratinocyte differentiation, keratinization, cornification, and interferon signalling. Functional classification of differentially expressed genes using PANTHER additionally implicated WNT signalling, EGFR signalling and inflammatory signalling pathways as dysregulated in KLICK syndrome. KLICK syndrome can therefore be characterized as a hyperproliferative inflammatory skin disorder. Using a reverse transcriptomics approach, epidermal growth factor receptor (EGFR) inhibition and phosphoinositide 3-kinase (PI3K) inhibition were identified as potential treatment options that warrant further investigation. Understanding the pathobiology of KLICK syndrome may yield novel mechanisms driving inflammatory skin disease, which may be therapeutically targetable in patients with currently limited treatment options.
P25 Hsa-miR-6727-5p contributes to the impact of high-density lipoprotein on fibroblast wound healing <i>in vitro</i>
Authors: Khaled Bastaki, Jamie Tarlton, Richard Lightbody, Patricia Martin, Annette Graham
Journal: British Journal of Dermatology (2023)

Abstract There is a clear unmet clinical need to develop effective strategies to improve chronic nonhealing wound closure rates, with distinct skin cell populations responding to potential therapies in different ways. Recent evidence suggests that high-density lipoproteins (HDL) can enhance wound closure rates, but, owing to the complexity, heterogeneity and relative instability of this lipoprotein fraction, developing a translational rationale to explore this observation is challenging. In the present work we explored the ability of primary human neonatal keratinocytes (HKn) and fibroblasts (HDFn), and adult dermal fibroblasts (HDFa), to respond to HDL by monitoring cholesterol efflux and scratch wound assays. Global changes in expression of small noncoding microRNA sequences (miRs), following the exposure of HDFn and HDFa to HDL, was determined by microchip array, validated by independent quantitative polymerase chain reaction, and the impact of a selected miRNA mimic on scrape wound closure assessed. All experiments were performed in triplicate. The data revealed that all cell types respond to HDL in terms of cholesterol efflux, confirming the functionality of this lipoprotein, but promotion of wound healing only occurs in HDFn and HDFa cells, with no response in HKn. Exposure of both HDFn and HDFa cells to HDL induces multiple changes in the expression of miR sequences, including hsa-miR-6727-5p. Levels of this miR increased in HDFn, but decreased in HDFa cells, following HDL exposure. Delivery of a hsa-miR6727-5p mimic repressed the expression of different target genes in HDFn (ZNF584) and HDFa (EDEM3, KRAS), while rates of wound closure were enhanced in the presence of a hsa-6727-5p mimic in HDFn cells and an inhibitor in HDFa cells. Intriguingly, analysis of the microarray data also revealed distinct expression landscapes in neonatal and adult fibroblasts, providing insight into the epigenetic evolution of these cells during the wound healing process. Thus, exposure to HDL has distinct impacts on the expression of the miR hsa-mir-6727-5p in neonatal and adult fibroblasts, which play differential roles in wound healing. We identify a range of pathways influenced by this miR but note that this miR alone cannot replicate the myriad effects of HDL. Future work will explore the impact of (dys)functional HDL and hsa-miR-6727-5p on wound closure rates in fibroblasts isolated from diabetic patients. Moreover, this study offers opportunities to expand knowledge of how profound changes in epigenetic profile can influence wound healing during ageing. Funding sources: K.B. was supported by a PhD scholarship provided by the Kuwaiti government; human fibroblasts were provided by the GCU Research Tissue Bank (NHS REC Ref 16/E0069).
When I use a word . . . Medical slang: neologisms
Authors: Jeffrey K Aronson
Journal: BMJ (2023)

Neologisms are words or phrases that are new to the language. As far as I know, there is no specific term that distinguishes neologisms that are derived from existing words from those that are completely newly minted. In the case of medical slang, only about 1% of terms are of the latter variety, not derived from pre-existing words. They include: the Amyoyo syndrome; gork, crumpo, and vedgy; Mudd-Fudd; smeck, smecker, and pisher; ganja, bindle, dujie, and mojo; langer, wazz, and chunder. Muggles and zatch, otherwise completely unrelated, by coincidence create a curious connection involving Louis Armstrong and J K Rowling.
P07 Optimizing DNA specificity and applicability of base and prime editors on <i>COL7A1</i> variants causing recessive dystrophic epidermolysis bullosa
Authors: Imogen Brooks, Maria Rudnytska, Ruta  Gebrezgabher, Lukasz Laczmanski, Gregory  Newby, David Liu, John McGrath, Joanna Jackow
Journal: British Journal of Dermatology (2023)

Abstract Base and prime editors are novel CRISPR variants that reduce the risk of off-target effects by avoiding double-stranded DNA cleavage, bypassing the need for homology-directed repair. These tools offer great therapeutic potential for inherited skin diseases such as recessive dystrophic epidermolysis bullosa (RDEB). Biallelic loss-of-function mutations in COL7A1 cause RDEB, resulting from loss of functional type VII collagen (C7) in the basement membrane zone. Current therapies for RDEB are limited, but ex vivo and in vivo COL7A1 base or prime editing therapies can potentially restore normal protein production with a single intervention treatment. This study compared DNA editing specificity, safety and efficiency on RDEB fibroblasts harbouring a homozygous c.4448 G&amp;gt;A (p.Gly1483Asp) mutation in COL7A1. Firstly, transfected adenine base editor (ABE8e) mRNA together with single guide RNA (sgRNA) were introduced into patient primary fibroblasts with Lipofectamine MessengerMax. By varying concentrations and ratios of guide RNA and ABE8e mRNA, we have achieved 100% biallelic correction without introducing any bystander mutations using 200 ng ABE8e and 28 ng sgRNA by transfecting 100 000 cells. Initial prime editing experiments are underway with PEmax plasmid and engineered prime editing guide RNAs (epegRNA) to optimize the guide design and to compare PE2max and PE4max editing systems. The functionality of the 100% biallelic correction using ABE8e is being evaluated with Western blot and in three-dimensional skin models. These early experiments demonstrate the potential value of base editing as a gene-editing tool in RDEB therapeutics; however, more functional work and off-target analyses are needed.
P03 Hydroxypyridone antifungals induce myofibroblast apoptosis in an <i>in vitro</i> model of hypertrophic scars
Authors: Alice Lapthorn, Marcus Ilg, Justine Sullivan, Peter Dziewulski, Selim Cellek
Journal: British Journal of Dermatology (2023)

Abstract Despite being the most common complication following burn injury, there are no medications to prevent the formation of hypertrophic scars. Myofibroblasts are key cells involved in scar formation, responsible for producing excessive amounts of extracellular matrix and have been shown to be resistant to apoptosis. We have previously shown that hydroxypyridone antifungals can inhibit the transformation of fibroblasts to myofibroblasts, and exhibit other antifibrotic effects. This study aimed to investigate the effect of these drugs on established myofibroblasts. Primary human dermal fibroblasts, derived from scars of patients with burns, were exposed to 10 ng mL–1 transforming growth factor (TGF)-β1 to induce myofibroblast transformation. The In-Cell enzyme-linked immunosorbent assay method was used to measure α-smooth muscle actin (α-SMA), with DRAQ5 staining used to measure cell viability. Concentration–response curves for the three hydroxypyridone antifungals (ciclopirox, ciclopirox ethanolamine and piroctone olamine) were constructed (0.03–300 µmol L–1) to see if the drugs could reverse myofibroblast transformation. TUNEL staining was used to investigate if the drugs (at 10 µmol L–1) induced apoptosis in the myofibroblasts. When applied to established myofibroblasts, the hydroxypyridone antifungals did not reverse myofibroblast transformation. Instead, the drugs exhibited a concentration-dependent decrease in myofibroblast cell viability. A 22% reduction in myofibroblast cell viability was observed at 10 µmol L–1, compared to fibroblasts treated in coincubation with TGF-β1 and the hydroxypyridones. To see if this reduction in cell viability was due to the induction of apoptosis in the myofibroblasts, TUNEL staining was used. TUNEL staining confirmed that the hydroxypyridone antifungals induced apoptosis in myofibroblasts after 72 h of treatment. This affect was not seen in the fibroblasts treated in coincubation with TGF-β1 and the hydroxypyridone antifungals. This is the first study to show that hydroxypyridone antifungals can induce apoptosis in established myofibroblasts, derived from burn scar tissue. Together with our previous results, this suggests that hydroxypyridone antifungals can prevent scar formation by tackling continuous myofibroblast transformation and myofibroblast persistence.
PostersP01 Tissue slice culture as a patient-derived explant model to investigate immunity and immunotherapy in cutaneous squamous cell carcinoma
Authors: George Coltart, Chester Lai, Aymen Al-Shamkhani, Eugene Healy
Journal: British Journal of Dermatology (2023)

Abstract Cutaneous squamous cell carcinoma (cSCC) is one of the commonest cancers worldwide with the capacity to metastasize. Immune evasion by the tumour prevents its elimination and allows it to progress, including enabling the cancer to spread to local lymph nodes and distant organs. Targeting this dysfunctional immunity is a key strategy for the treatment of cSCC and anti-PD-1 checkpoint inhibition is now the first-line therapy for advanced and metastatic cSCC. However, complete responses to anti-PD-1 are seen in &amp;lt; 15% of patients and therefore it is vital that other strategies for enhancing anticancer immunity in cSCC are explored. Given the expense of clinical trials, it is essential to have good preclinical data to target cancer drug development appropriately, and this can be challenging in the investigation of cancer immunotherapy, as these treatments rely upon an intact human immune–tumour interface. We have developed a tumour slice culture (TSC) system to investigate the mechanism and efficacy of immune-modulating anticancer therapy in cSCC. This patient-derived explant model involves taking tissue slices from freshly excised tumours and culturing the slices under different experimental conditions, while maintaining the tumour microarchitecture of each slice. We cultured &amp;gt; 50 tumours using the TSC platform, including 35 cSCCs. Immunofluorescent confocal microscopy of anti-CD4 demonstrates penetration of antibody throughout the tissue slice. The mean (SD) viability of lymphocytes (T and B cells) determined by flow cytometry after 72 h of culture was 90.3% (7.89%; n = 31). Treatment of slices with OKT3 anti-CD3 antibody for 72 h induces a dose–response increase in T-cell Ki67 expression (2.87% unstimulated vs. 13% OKT3 100 ng mL–1; P &amp;lt; 0.001) and CD8+ T-cell granzyme B (47.6% unstimulated vs. 63.7% OKT3 ng mL–1; P = 0.064), as detected by flow cytometry (n = 7). Enzyme-linked immunosorbent assay of the culture supernatant showed that interferon-γ concentrations increased from &amp;lt;9.6 pg mL–1 (unstimulated) to 131.1 pg mL–1 [OKT3 1000 ng mL–1 (n = 6, P &amp;lt; 0.05]. To our knowledge the TSC system has not previously been used in cSCC and represents a promising technique to investigate new potential immunotherapies in this cancer.
O21 Single-cell RNA sequencing and proteomic analysis of dermal fibroblasts cultured from female scalp identifies significant alterations in cell populations with age
Authors: Richard Baker, Rachael Williams, Gillian  Westgate, Alison Pawlus, Jeanna Zguris, Julie Thornton
Journal: British Journal of Dermatology (2023)

Abstract We have previously reported that significant in situ detrimental changes occur in the female scalp with age. We also reported that primary cultures of female scalp dermal fibroblasts (DF) significantly increase the secretion of proteins such as cartilage oligomeric matrix protein/thrombospondin-5 (COMP/TSP5), secreted protein acidic and cysteine rich (SPARC) and smooth muscle actin (ACTA2). This highlights changes in DF cellular identity with age, altering the immediate hair follicle environment, presumably driving hair ageing. To gain a better understanding of age-related molecular and functional changes in DFs, we compared both the transcriptional and proteomic profile of primary cultures of scalp DFs (passage &amp;lt; 3) from female donors (n = 8) aged 21–77 years. Transcriptional profiles were analysed via 10× Genomics scRNAseq. Data profiles were generated by evaluation of cellular lysates by liquid chromatography mass spectrometry proteomics and compared by MS1 precursor intensities using PEAKS software. A total of 16 166 DF cells from young (n = 3) and older (n = 3) donors were sequenced. Across the two populations, 11 transcriptionally distinct clusters were detected, highlighting the heterogeneity in DFs. The percentage of cells in these clusters changed with age, and gene set enrichment analysis ascertained a significant increase of cells in cluster 2, which represented a higher number of gene sets related to fibrosis, senescence, collagen synthesis and degradation. Proteomic analysis of DF cellular proteins (four donors in each age group) identified 11 significantly differentially expressed proteins between the two age groups. Two proteins (serine/threonine-protein kinase 38 and rho GTPase activating protein 21) were significantly reduced in the older cohort, while nine proteins (smoothelin, retinol dehydrogenase 5 tetranectin, oestradiol 17-beta-dehydrogenase 11, CD14, carboxypeptidase M, gamma-enolase, plexin-B1 and endoplasmic reticulum membrane protein complex subunit 4) were significantly elevated in the older cohort. Gene Ontology and STRING analysis of the data revealed modifications to key pathways with age (e.g. collagen and elastic fibre formation, autophagy, ubiquitination and metabolic markers). This study highlights the loss of scalp DF cell signature and identity with age. We hypothesize that these changes impact on the ability of the scalp dermal environment to support and maintain healthy hair follicles, contributing to the age-related changes observed in female hair growth.
Randomized-controlled trial assessing a digital care program versus conventional physiotherapy for chronic low back pain
Authors: Di Cui, Dora Janela, Fabíola Costa, Maria Molinos, Anabela C. Areias, Robert G. Moulder, Justin K. Scheer, Virgílio Bento, Steven P. Cohen, Vijay Yanamadala, Fernando Dias Correia
Journal: npj Digital Medicine (2023)

Low back pain (LBP) is the world's leading cause of years lived with disability. Digital exercise-based interventions have shown great potential in the management of musculoskeletal conditions, promoting access and easing the economic burden. However, evidence of their effectiveness for chronic LBP (CLBP) management compared to in-person physiotherapy has yet to be unequivocally established. This randomized controlled trial (RCT) aims to compare the clinical outcomes of patients with CLBP following a digital intervention versus evidence-based in-person physiotherapy. Our results demonstrate that patient satisfaction and adherence were high and similar between groups, although a significantly lower dropout rate is observed in the digital group (11/70, 15.7% versus 24/70, 34.3% in the conventional group; P = 0.019). Both groups experience significant improvements in disability (primary outcome), with no differences between groups in change from baseline (median difference: -0.55, 95% CI: -2.42 to 5.81, P = 0.412) or program-end scores (-1.05, 95% CI: -4.14 to 6.37; P = 0.671). Likewise, no significant differences between groups are found for secondary outcomes (namely pain, anxiety, depression, and overall productivity impairment). This RCT demonstrates that a remote digital intervention for CLBP can promote the same levels of recovery as evidence-based in-person physiotherapy, being a potential avenue to ease the burden of CLBP.